Endemic (Balkan) nephropathy (EN) is a devastating disease affecting men and women living in rural areas within the Danube river basin, encompassing regions of Croatia, Bulgaria, Romania, Serbia and Bosnia. The clinical course of EN is characterized by an insidious onset, invariable progression to chronic renal failure and a strong association with urothelial cancer. Significant epidemiologic features of EN include its occurrence within certain villages; among these villages, only members of certain households are affected. It is generally believed that EN is an environmental disease related to the farm life style in endemic regions. Hypotheses have been advanced over many years to explain the etiology of EN; we are pursuing an earlier, largely ignored and untested hypothesis that seeds of Aristolochia clemantitis contaminate grain used in baking bread, a dietary staple in the endemic region. This hypothesis is supported by striking similarities between the pathophysiology of EN and the recently recognized syndrome of aristolochic acid nephropathy. Scientists based at the Univ. of Zagreb School of Medicine and Rudjer Boskovic Inst. (Zagreb), together with physicians responsible for the clinical care of patients with EN (Slavonski Brod), propose to test the aristolochic acid etiology hypothesis by conducting a molecular epidemiologic, case-control study involving 750 adult residents of three endemic villages. Residents (150) of a nearby nonendemic village serve as controls. Croatian scientists will conduct analytical studies designed to quantify the hallmark biomarker predicted by the guiding hypothesis; namely, DNA-aristolactam adducts in renal tissues and exfololiated urothelial cells in urine of patients with EN. In addition, the spectra of P53 mutations in urothelial tumor tissues obtained from long-term residents of the endemic region will be established. If our hypothesis proves to be correct, public health measures could be instituted that would lead to the eradication of EN in Croatia and throughout the Balkan region, where 100,000 persons are currently at risk. To advance that goal, we propose to initiate, in parallel, pilot epidemiologic studies in the endemic regions of Bosnia and Bulgaria where current exposure will be monitored by measuring the aristolochic acid content of flour. This FIRCA will be conducted almost entirely in Croatia, in collaboration with Dr. Bojan Jelakovic, lead foreign investigator (University of Zagreb School of Medicine) with P01 ES04068 serving as the parent grant. [unreadable] [unreadable]